Based on network pharmacology and molecular docking to predict the mechanism of TMDZ capsule in the treatment of IS

Background: Ischemic stroke (IS) is the most common cardiovascular and cerebrovascular disease in clinic. Qiangli Tianma Duzhong Capsule (TMDZ capsule) has significant therapeutic effect to IS. Therefore, it is great significance to explore the mechanism of action of TMDZ capsules in the treatment of IS. Methods: The potential active components and possible targets of TMDZ capsule were obtained from TCMSP and The Encyclopedia of Traditional Chinese Medicine databases. IS related targets were collected by Genecard database, OMIM database, TTD database and DisGeNET database. The common target network of drug-diseases was constructed using Cytoscape for visualization analysis. Potential mechanisms were identified through enrichment analysis of gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Three key targets (ALB, TNF, and INS) were selected from the key networks with high correlation scores in PPI for molecular docking, through molecular docking, the interaction between target and protein is visualized. Results: 59 active components and 648 targets of TMDZ capsules and 2286 targets of IS were obtained through database mining. Compound-target network is constructed with 117 nodes and 1185 edges. GO and KEGG suggest that lipids and atherosclerosis, fluid shear forces and atherosclerosis, neurodegenerative pathways – multiple diseases and blood circulation play important roles in the treatment of IS. Conclusions: This study reveals the molecular mechanism of TMDZ capsules in the treatment of IS by integrating molecular docking with a network pharmacological strategy, which not only confirmed the clinical efficacy of TMDZ capsule, but also laid the foundation for further experimental research.


Introduction
Stroke is the most common cardiovascular and cerebrovascular disease in clinic [1] and also known as cerebrovascular accident, it is the second leading cause of death and the third leading cause of disability in the world. [2] Its 2 major categories are ischemic stroke (IS) and hemorrhagic stroke. [3] About 18 million people suffered from IS in 2020, with two-thirds of IS occurring in people over 65 years of age, [4,5] and South Asians are higher risk of developing IS particularly, accounting for 40% of global IS deaths, it imposes an enormous burden on health care systems worldwide. IS accounts for 70% to 80% of stroke, and the research on effective treatment of IS is one of the hot spots of stroke at present. [6,7] IS is characterized by early absence of obvious symptoms and high recurrence rate, it is easy to develop into cerebral infarction with the gradual aggravation of the disease. [8] With the clinical gradual attention of traditional Chinese medicine (TCM) The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. treatment of diseases and Chinese medicine has also made positive progress in the treatment of cardiovascular and cerebrovascular diseases, so we should actively adopt the method of integrated traditional Chinese and western medicine for clinical treatment. [2,9] The common pathogenic factors of IS are arterial thrombosis and venous thrombosis, which are distinguished by embolism of attached blood vessels, arterial thrombosis is associated with a high risk of IS. In view of the behavioral disorders caused by IS, TCM put forward the concept of "qi deficiency and blood stasis." [6,10,11] From the TCM for the treatment of IS, it was found that the Qiangli Tianma Duzhong Capsule (TMDZ capsule) contained part of the TCM for the treatment of IS, According to the investigation, the TMDZ capsule with TCM ingredients has obvious clinical efficacy in the treatment of cardiovascular and cerebrovascular diseases. [6] At present, TMDZ capsule has been registered in the national medical insurance, in clinical practice, it is mainly used for the symptoms of meridian pain, limb numbness, inconvenient walking, waist and leg pain, headache and dizziness caused by stroke. TMDZ capsules can improve microcirculation, slow heart rate, prolong ejection time, increase cerebral blood flow, reduce myocardial oxygen consumption, have good sedative and hypnotic effects, and reduce sympathetic nerve excitability, these effects have certain therapeutic on related symptoms caused by IS. [12] TMDZ capsule is a multi-target drug with good efficacy in the treatment of IS, the network pharmacology method can explain the characteristics of "multi-component and multi-target" of TMDZ capsule, which is consistent with the complex etiology and multi target treatment concept of IS. [13,14] In this study, network pharmacology was adopted to predict the active ingredients, targets and pathways of TMDZ capsules, the specific steps are shown in Figure 1. Finally, key targets of TMDZ capsules in the treatment of IS were located in the integrated gene expression dataset, which could evaluate the corresponding relationship and explain the mechanism of action of TMDZ capsules in the treatment of IS. It could provide a new strategy in the treatment of IS.
Because the use of the ETCM and TCMSP to predict the potential compounds and target of TMDZ capsules has limitations, we searched the extensive literature through the Wanfang database, CNKI, Chaoxing, PubMed and other databases to supplement the relevant data. Then targets were entered into the Uniport database for target normalization.
All targets were standardized using the Uniprot database and the species should be selected as "Homo snpiens" when using the Uniprot database for target and Gene name conversion.

Screening of common targets of drug components and diseases
The core targets of TMDZ capsules in the treatment of IS were obtained by intersecting the targets of the active chemical components in TMDZ capsules with the genes related to IS disease and the Venn diagram was prepared, which requires the use of the Venny online platform (https://bioinfogp.cnb.csic.es/tools/ venny_old/). [21] 2.4. The network diagram of "active ingredient-target" In order to elucidate the relationship between the active chemical constituents and targets, the composition-target diagram of drug was constructed by Cytoscape3.8.2. Important network topological parameters of the compound and related targets were calculated, such as degree, intermediate centrality, and proximity centrality. the larger the node and node connectivity, which related to the more functions in the network diagram.

Construction of PPI network diagram and screening of core targets of TMDZ capsules in the treatment of IS
Protein-protein interaction (PPI) network diagrams are constructed to illustrate the interactions of proteins and interactions between proteins and other molecules, which can predict intracellular effects. The core target of TMDZ capsule treatment IS entered into the String online platform (http://string-db. org/) [22] for PPI, which can explain the therapeutic mechanism of action of TMDZ capsule more comprehensively. In order to meet the accuracy and quantity requirements of PPI, the confidence score was selected to be greater than 0.4, the species was limited to Homo sapiens, then PPI data was obtained. The results are saved in tsv format. PPI results are imported into Cytoscape3.8.2 software to draw the interactive network, then core targets were selected by double degree value.

Analysis of target function and pathway enrichment in the treatment of IS with TMDZ capsule
To further clarify relevant functions and pathways, gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out by the metascape platform (http://metascape.prg/gp/ index.html), [23] results were obtained with human was defined as the species. After getting the results, these results are visualized as GO term figure and bubble chart based on bio-informatics platform (www.bio-informatics.com.cn). metascape platform is a powerful gene functional database, which can provide comprehensive functional annotations of pathways, including biological processes, cellular component and molecular functions.

Molecular docking validation
Degree of nodes is a key indicator to describe network nodes. The top 3 core targets of degree values (ALB, TNF, and INS) were selected from PPI networks. The corresponding chemical components of p-hydroxybenzaldehyde, carotenoside and palmitic acid were found, then Molecular docking of the target and the corresponding chemical components was performed. The protein structure of the key target was obtained from the PDB database (https://www.rcsb.org/), [24] the chemical structure of the 3 components was drawn by ChemDraw18.1. The prepared protein structure and molecular structure were imported into PyMOL to remove water molecules and small molecule ligands, the AutoDockTools software was used to convert the protein and drug components into pdqt format files and identify active pockets, finally, molecular docking was performed by vina software. The greater the absolute value of binding energy, the stronger the binding ability of the compound and the protein target.

Active compounds and putative targets of TMDZ capsule
The active compounds of the 12 TCM were obtained in TMDZ capsules from the ETCM database and TCMSP database. A total of 118 active components were obtained, 12 components from Gastrodia elata, 28

Putative targets of IS
A total of 2334 targets were identified, 2144 targets were identified in Genecard database, 129 targets were identified in OMIM database, 61 targets were identified in DrugBank database and 0 targets were identified in the therapeutic target database database. 2286 targets were defined after removing 48 duplicate targets. Medicine

Analysis of "active ingredient-target" network diagram
Through Venny online platform, 130 common targets of drugs and diseases were obtained, as shown in Figure 2. "active ingredient-target" network diagram of TMDZ capsules is shown in Figure 3. which can obtain 127 nodes and 1185 edges. it shows that Gastrodia elata and eucommia ulmoides have more relevant components and targets, while Cyathula officinalis and Radix Aconiti Kusnezoffii have less relevant components and targets. the node of higher degree can connect more edge, which indicate that the node is important in the network, so CASP3 is associated with a variety of herbs in TMDZ capsules, which indicate that CASP3 is important.

Construction of PPI network diagram and screening of core targets
Protein interaction relationship data and protein interaction network diagram were obtained, as shown in Figure 3. 127 nodes and 1185 edges were obtained. 25 nodes and 268 edges were obtained after screening key targets. 18 key targets were obtained through PPI network screening, the result is shown in Figure 4. In the PPI network, the darker the color and the larger the node area, which indicate the node is more important, among them, the top 10 targets with high degree value were albumin (ALB), tumor necrosis factor (TNF), insulin (INS), cell tumor antigen p53 (TP53), caspase 3 (CASP3), Myelocytomatosis (MYC), Catenin Beta-1 (CTNNB1), Vascular Endothelial growth factor A (VEGFA), sparse representation-based classifier (SRC), peroxisome proliferative activated receptor γ (PPARG), prostaglandin proliferative peroxidase synthase 2 (PTGS2).

Functional and pathway enrichment analysis of key targets
In order to further explore the mechanism of action of TMDZ capsule treatment of IS, potential therapeutic targets were analyzed for GO and KEGG enrichment (the top 10 results of each analysis are shown in Table 1). GO functional enrichment analysis showed that GO entries included 470 biological processes (BP), 72 cellular components (CC), and 133 molecular functions (MF). The first 10 entries of −lgP values of BP, CC and MF are  shown in Figure 5. A total of 141 KEGG pathway enrichment analyses were obtained. According to the −lgP value, top 10 items is shown in Table 1 and the bubble plot of top 20 items is shown in Figure 6.
Through GO functional enrichment analysis and KEGG pathway analysis, ion channel complex, lipid binding, blood circulation, lipids and atherosclerosis, fluid shear stress and atherosclerosis, pathways of neurodegeneration multiple diseases and other processes were related to the treatment of IS with TMDZ capsule. Through the above process, we can speculate the mechanism of action of TMDZ capsule in the treatment of IS.

Molecular docking of active compounds
Molecular docking simulates the interaction between small molecular ligands and bio-macromolecule receptors, which can predict the binding mode and affinity between them and selects the lead drug of the target, it could aid drug development. In this study, the top 3 targets with the highest degree value were selected for molecular docking, the results were shown in Figure 7. According to the Figure 7, the target of smaller binding energy had stronger binding affinity with the target protein.
The binding energy of ALB, INS, and TNF was −6.3, −6.49, and −6.67 kcal/mol, so the target could easily bind its corresponding protein. These results can indirectly confirm the regulatory effect of TMDZ capsules on IS targets. At the same time, the above molecular docking results are consistent with the previous network screening results.

Category
Through molecular docking, it can be seen that p-hydroxybenzaldehyde, carotoside and palmitic acid molecules are closely connected with the targets of ALB, TNF and INS, which can be inferred that the main drugs in the TMDZ capsule are gastrodia and eumoides. Its main components play a role in the prevention and treatment of IS by reducing the level of EAA neurotransmitter Glu, [48] promoting endothelial cells to release neurotrophic factors VEGF-A and BDNF, [49] enhancing nerve cell to resistance damage, [50] promoting the proliferation of neural stem cells and regulating cholesterol levels. [51,52] This role is mainly related to lipids, ion channels, atherosclerosis pathways and neurodegeneration pathways. The results showed that TMDZ capsules had the effects of anti-oxidation, reducing blood cholesterol, inhibiting cerebral arteriosclerosis, inhibiting thrombosis, activating platelets and reducing the release of platelet inflammatory factors. Furthermore, the mechanism of action of TMDZ capsules was further explained in the prevention and treatment of IS.

Conclusion
This study systematically explored the active ingredients of TMDZ capsules and the potential targets and signaling pathways of TMDZ capsules in the treatment of IS. The results showed that the TMDZ capsule was composed of 12 drugs, a total of 59 active compounds and 648 targets. There were 2286 targets for IS, and 130 targets for the common action of TMDZ capsules and IS. TMDZ capsule mainly acts through vanillin, p-hydroxybenzaldehyde, palmitic acid, carotene, quercetin, β-carotene and other components in the treatment of IS. The corresponding targets of these components are ALB, TNF, INS, and so on. It mainly plays a role through lipid and atherosclerosis, fluid shear stress and atherosclerosis, neurodegenerative pathway-multiple diseases and other pathways. In addition, molecular docking successfully verified the tight connection between the active compounds and the IS targets. This study explored the mechanism of action of TMDZ capsules in the treatment of IS by network pharmacology, which provided some reference for the clinical use of TMDZ capsules in the treatment of IS.